Presently, a patient's red cell concentration in the blood, which is the net of production-loss, is monitored by measuring the hematocrit count in the patient's blood. The hematocrit is the percentage volume that packed red blood cells occupy in a centrifuged sample of whole blood. Hematocrit determinations may presently be performed by filling a small bore glass tube with anticoagulated whole blood, sealing one end of the tube, and centrifuging the tube to pack the red blood cells. After packing, which takes about three to five minutes in a small centrifuge, the length of the packed red blood cell column and the total filled length are measured, and the hematocrit, expressed as a percentage, is calculated. The hematocrit determination gives no information as to red cell production and/or loss.
U.S. Pat. Nos. 4,027,660; 4,181,609; 4,156,570; 4,558,947; and others describe a procedure which involves drawing a sample of anticoagulated whole blood into a capillary tube, placing a float in the tube with the blood sample, and centrifuging the blood sample to cause the float to settle into the red cell layer, and allow differential buffy coat constituent counts to be measured. This technique is also used to measure hematocrit by applying a correction factor to account for the sinking of the float into the packed red cells, and can account for the shrinking of the packed red cells by any additives in the blood sample, such as potassium oxalate, if present. The hematocrit count is a reliable procedure for measuring the red cell content of the blood at the time the measurement is made, but it may not detect certain conditions relating to production of new red blood cells, or the loss of red blood cells. If the hematocrit is low, the physician will be alerted to the fact that a current condition exists which is either supressing new red blood cell formation; or that an atypical loss of red blood cells has occurred or is ongoing with insufficient increased production of red cells. Hematocrit determinations cannot, however, detect such atypical conditions unless they are relatively concurrent with the time the hematocrit is read. Thus, for example, if a patient has experienced a bleeding episode three weeks before the hematocrit is read, the blood test will not necessarily reveal the episode since the loss of blood may have caused increased red cell production to compensate for the bleed.
Red cells that are one day old maintain fragments of nucleic acid which, when stained with new methylene blue, cause them to appear as reticulocytes. By staining a smear of red blood cells, and then manually counting the percentage of red cells that are reticulocyes, one can estimate the present red cell production rate. Alternatively, reticulocytes may be enumerated in a blood sample with commercially available stains wherein the cells are detected and counted by fluorescent activated cell sorters (FACS). Neither the normal stain and smear procedure nor the FACS will be able to detect, for example: a red cell production rate that is five times the normal rate; which lasted for three days; and which increased production took place ten days prior to the blood sampling. These prior art procedures can detect increased or decreased production episodes only during the episodes, or for one day thereafter.
L. M. Corash et al, in the July, 1974 issue of The Journal of Clinical Medicine describe a procedure for separating erythrocytes according to age on a simplified density gradient. This publication states that age-dependent separation of erythrocytes may be accomplished by a variety of prior art cell supporting substances such as: bovine serum albumin; phthalate esters; dextran; and gum acacia. The publication states that these prior art procedures are undesirable and suggests that an arabino galactan polysaccharide of 30,000 daltons be used in lieu of the prior art supporting substances. The Corash et al procedure however requires the use of defibrinated, washed, and packed human erythrocytes which are layered on top of a performed medium gradient.
The tube containing the packed cells and medium is filled with mineral oil and then centrifuged to obtain the separation of cells. Thus the Corash et al procedure is time consuming and complicated.